Production of double-stranded ribonucleic acid

ABSTRACT

DOUBLE-STRANDED RIBONUCLEIC ACID IS PRODUCED BY ESCHERICHIA COLI CELLS INFECTED WITH THE MU-9 MUTANT OF MS2 COLIPHAGE AND THE SPECIFIC PRODUCTIVITY IS INCREASED BY THE TIMELY ADDITION OF AN ANTIBILTIC POST-INFECTION.

United States Patent 3,582,469 PRODUCTION OF DOUBLE-STRANDED RIBONUCLEIC ACID Jerome Birnbaum, Morganville, N.J., assignor to Merck & Co., Inc., Rahway, NJ. No Drawing. Filed Sept. 27, 1968, Ser. No. 763,408 Int. Cl. C12d 13/06 US. Cl. 195-28 6 Claims ABSTRACT OF THE DISCLOSURE Double-stranded ribonucleic acid is produced by Escherichia coli cells infected with the MU-9 mutant of M52 coliphage and the specific productivity is increased by the timely addition of an antibiotic post-infection.

RELATED CASES Ser No. 744,687 filed July 15, 1968 entitled Production of Interferon Inducers by Barbara D. Lago.

This invention relates to the production of interferon inducers and particularly to a process for improving the yield of double-stranded ribonucleic acid produced by Escherichia coli cells infected with the MU-9 mutant of MS2 coliphage.

The fact that double-stranded ribonucleic acid may come from this source is stated in an article entitled, Inducers of Interferon and Host Resistance, IV. Doublestranded Replicative Form RNA (MS2-RF-RNA) from E. coli infected with MS2-coliphage, by A. K. Field, G. P. Lampson, A. A. Tytell and M. R. Hilleman, appearing in the Proceedings of the National Acadamy of Sciences (U.S.), vol. 58, No. 5 (November 1967), pp. 2102-2108. In that article it is reported the MS2 coliphage when grown in E. coli cells produces a double-stranded ribonucleic acid which proves to be an excellent inducer of interferon when administered to animals. This is also true of the MU-9 mutant which is obtainable from the New York University School of Medicine and this invention is concerned with this variant.

The above journal article describes the manner of E. coli culture, its infection with MSZ coliphage, and the recovery of the double-stranded ribonucleic acid (hereinafter called DSRNA) in a purified form. Recent advances in this art made by Barbara D. Lago as disclosed and claimed in her patent application, Ser. No. 744,687, filed on July 15, 1968 have shown that the growth of the E. coli in a corn steep liquor medium and infection With the MU-9 mutant of MS2 coliphage, produces an increase of up to ten times and even more as much cell growth and a correspondingly greater amount of DSRNA. It is an object of the present invention to additionally increase the production of DSRNA from the E. coli culture which has been infected with the MU-9 mutant of M82 coliphage.

In accordance with the present invention it has been found that the specific productivity of the DSRNA relative to the amount of E. coli can be increased. This means that a greater amount of DSRNA can be obtained from the same number of E. coli cells or alternatively that the same amount of DSRNA is produced by fewer E. coli cells than were formerly required. This has the additional advantage of facilitating isolation of the DSRNA because of its greater concentration in the growth medium.

In practicing this invention the E. coli can be grown in any one of the known culture media which will support its growth, and representative of these are media containing tryptone and/or corn steep liquor. To obtain more eificient use of the culture medium the E. coli should first be permitted to attain substantial growth, namely in excess 3,582,469 Patented June 1, 1971 of 5x10 cells per ml. of broth and preferably 5x10 cells per ml. but a greater number of cells can be utilized. At this time the MU-9 is added so that there are at least three to thirty but preferably six infective virus particles per single bacterium. Growth is continued so that the E. coli becomes fully infected.

The present invention increases the specific productivity (mg. DS-RNA/ g. dry cells) by inhibiting the growth of E. coli (after infection with the virus) without affecting the level of DSRNA produced. This is accomplished by adding growth-inhibitory antibiotics to the fermentation broth at a specified time after infection. The actual mechanism of increased specific productivity is to arrest E. Coli cell growth in the tryptone medium without affecting synthesis of DSRNA. Furthermore, in corn steep liquor medium there was a direct stimulation of DSRNA synthesis coincident with a reduction in cell growth.

The antibiotics which are the most satisfactory to use in practicing the invention are the ones which inhibit protein synthesis such as, streptomycin, oxytetracycline and chloramphenicol but the invention is not limited to these specific antibiotics as the invention includes other bactericidal agents elaborated by the fermentative growth of microorganisms. They are added to the growth medium at 45 to 120 minutes post-infection and in the amount of 5 to 200 micrograms, preferably 50 to micrograms, per ml. of the growth culture. The antibiotic may be added a few minutes earlier than 45 minutes or a few minutes later than minutes but with diminished production of the DSRNA relative to the E. coli, and generally the antibiotic should be added before 90 minutes post-infection time.

Although best results seem to occur if the culture medium is the corn steep liquor disclosed in said Lago application, the increased specific productivity of the invention is achieved also with commonly known culture mediums such as those containing tryptone. The increased specific productivity appears to be independent of the particular culture medium, but it would appear that with corn steep liquor it is obtained with or without growth inhibitor of the E. coli cells. With tryptone and like media the increased specific productivity is obtained only when the growth of E. coli is inhibited.

An additional benefit obtained from the antibiotic addition is that it decreases the likelihood of reversion of the mutant MU-9 back to MS2. During the prior practices of DSRNA fermentation, some of the mutant virus MU- 9 reverts back to the wild-type virus MS2. This reversion is undesirable since it may lead to lysis of the E. coli cells and cause loss of some of the DSRNA into the medium. The use of an antibiotic in both tryptone and corn steep liquor media hinders the development of the undesirable MS2 phage. Thus, the use of the antibiotic has the additional advantage of decreasing the development of revertants.

The recovery process may be the same as that outlined in said Lago application or in the aforementioned journal article by Field et a1. As is mentioned above, this recovery is facilitated because there will be a greater amount of the DSRNA relative to the E. coli cells and the growth medium.

Representative examples are the following;

Example I E. coli was grown and maintained on tryptone agar of the following composition in grams per liter of water: tryptone, 10; yeast extract 1.0; dextrose, 1.0; sodium chloride, 8.0; calcium chloride, 0.22; and agar, 15. Inoculum cells were prepared by growth for 16 hours at 37 C. in the same medium lacking agar. These cells were used to inoculate several identical batches of the tryptone 4 culture medium and incubated at 37 C. on a g'yrotary trates that the antibiotic was increasing specific proshaking device to aerate the growing cells. When the ductivity in the tryptone broth by inhibiting growth withpopulation reached 5x1 cells per ml. of broth, MU-9 out affecting DS-RNA synthesis. virus was added so that there were 6 infective virus particles per single bacterium. Incubation was then con- Example n tinned at 37 C. The procedure of Example I was carried out, using At various times post infection chloramphenicol was oxytetracycline; similar results are obtained as is shown added (except to the control batch) and after 300 minby Table 2.

. TABLE 2 Oxytetracycline added at 60 ng/ml.

Growth (dry weight) Double-stranded RNA Time of addition of oxytetracycline Percent of Percent of Mg./g., Percent oi post-infection, min. MgJrnl. control MgJl. control cells control Not added (control). 0. 54 100 39. 7 100 73. 5 100 0 0. 37 0.7 2 4. 0 5 0. 28 52 4. 2 11 15. 0 20 0. 36 67 25. 1 6 69. 8 95 0. 42 78 39. 7 100 90.0 122 0. 45 83 49. 0 123 109 148 0. 44 81 40. 0 100 90. 9 125 0. 43 80 35. 5 89 85. 0 113 utes post-infection each batch was assayed for DS-RNA Example 11 relativ to th dr Wei ht of the E. 01' cells. The results are shgwn g 1 g c l The procedure of Example I was carried out, uslng TABLE 1 Chloramphenicol added at 50 pig/ml.

Growth (dry weight) Double-stranded RNA Time oi addition of chloramphenicol Percent of Percent of Percent of post-infection, min; Mg./m1. control MgJl control MgJg: control Not added (control). 0. 52 100 49. 5 100 95. 2 100 Chloramphenicol added at 100 ngJml:

This Table 1 shows that when the antibiotic was added streptomycin. Similar results are obtained as is shown at 0 to 30 minutes after infection, there was a significant by Table 3.

TABLE 3 Streptomycin added at 50 ngJml;

Growth (dry weight) Double-stranded RNA Time of addition of streptomycin post- Percent 0! Percent cl MgJgi Percent of infection, min. MgJml; control MgJi; control ce control Not added (contr0l). 0. 67 100 41.4 100 61. 8 100 0 0.23 34 2. 52 6 9.0 15 0.34 51 3.10 s 9.1 15 0. 3a 52 e. 52 16 1s. 1 30 0. 40 69 27.6 01 60.0 91 0. 52 78 4e. 2 112 89. 0 14s 0. 56 84 42. 7 103 76. 4 122 0. 5e 84. 42. 7 103 76. 4. 122

inhibition of growth; however, there occurred also a Example 1V severe inhibition of DS-RNA synthesis. If the antibiotic was still inhibited but DS-RNA synthesis (mg/1.) was E. coli was added to each of several identical batches unaifected. However, the specific productivity (mg./g. of 00m Steep q r Prepared as follows:

dry cells) increased 45 to 54% in one experiment and RaWCOIH Steep q was dlluted Wlllh an 1 31 to 36% i th oth I tryptone, b th th t t l ume of water and solid sodium hydroxide is added until DS RNA synthesized (mg,/l i fla k receiving th pH 7 was attained. This solution 'was heated for 1 hour antibiotic were equivalent within experimental error, to at 100 C., cooled, centrifuged to remove insoluble mathat synthesized by untreated control cells. This illusterial, diluted 10 fold with water, heated to boiling, cooled and recentrifuged to remove any further insoluble material. The final supernatant fluid is used as growth medium for E. coli. E. coli cells are inoculated into this medium and allowed to incubate at 37 C. under shaking conditions.

At the time that 1.28 mg. (dry weight) of cells per ml. of culture was attained MU-9 bacteriophage was added to give 6 infective virus particles per bacterium, and incubation was continued. At various times, post-infection streptomycin was added (except to the control batch) and after 300 minutes post-infection each batch was assayed for DS -RNA relative to the dry weight of the E. coli cells. The results are shown in Table 4.

TABLE 4 increases productivity by either limiting growth after infection or by stimulating directly the synthesis of DS-RNA.

Example VI E. coli infected with MU-9 bacteriophage Streptomycin added at 100 pg/ml.

Growth (dry weight) Double-stranded RNA Time of addition of streptomycin post- Percent Percent Mg./g., Percent infection, min. MgJml. of control Mg./l. of control cells of control Not added (control).. 2. 66 100 127 100 47. 8 100 1.22 46 0 0 0 0 2.21 83 o 0 0 0 2.60 as 0 0 0 0 1.79 67 7.8 6 4.4 '10 2. 24 84. 14. 9 12 6. 7 14 2. 56 96 187 147 73. 153 2. 72 102 103 152 70. 9 148 As in tryptone medium the addition of the antibiotic TABLE 6 early in the infection inhlbited growth and DS'RNA The efiect olstreptomycin and time ofharvest on double-stranded RNA synthesis. When added at 90 to 120 minutes, there was a Synthesis by @011 infected with bacteriophage 48 to 53% increase in specific productivity (mg./g.). In gam a; strepigfiyqin Doubknsmnded RNA arves a H1111. Table 4, however, the increase was due to a direct effect posh postdnfectiony Percent of MgJg. Percent o of streptomycin on DS-RNA synthesis and not on growth infection, mln, 'pg./ ml. Mg./l. control cells control slnce the dry welght of the cells at harvest was equal to 180 100 242 109 mi 9 136 that of the control values. 180 (control) 223 100 71.0 100 300--. 100 208 118 93.0 141 Example V 800 0 (control).... 177 100 65.9 100 To determine the relative effect of various amounts of Table 6 shows that there is no need to wait 300 minstreptomycin the procedure of Example IV was carried utes before harvesting; 180 minutes after infection apout by adding the streptomycin to identical batches all at minutes post-infection. The results of the varying amounts of streptomycin on DS-RNA production are shown in Table 5. I

TABLE 5 pears to give somewhat better results. In this experiment, there was only a slight (9-1-8%) direct stimulation on DS-RNA synthesis and a 36 to 41% increase in specific productivity. A harvest time of 180 minutes seemed bet- Etlect of streptomycin on growth and double-stranded RNA synthesis by E. coli infected with MU-9 bacteriophage Growth (dry weight) Double-stranded RNA Streptomycin at 90 min. Percent Percent Mg./g., Percent post-infection, agJml. Mg./ml. of control Mg.ll. of control cells of control ter than 3 00 minutes because the total DS-RNA synthesized (mg/l.) was higher at the earlier period.

Examples VII and VIII The fact that other antibiotics can be used to obtain increased DS-RNA production is shown in the following Tables 7 and 8.

' TABLE 7 [Effect of penicillin on grolwlth and double-Stranded RNA synthesis by E. coli infected with U-9 bacteriophage in tryptone broth] Penicillin added at 100 a/gml.

(d i ilt we Do b t Time (it; adgitim}; of ry g u 1e s randed RNA penici n pos Percent of Percent of m Perc t of lnfeetlon, minutes MgJml. control Mg./l. control c ei GO I I tXOI Not added (control). 0. 58 100 36. 2 100 62. 100 0. 37 64 18. 8 52 50. 8 81 0. 42 72 18. 8 52 44. 8 72 0. 45 78 18. 3 51 40. 7 65 0. 50 86 33. 7 93 67. 4 108 0. 51 88 32. 8 91 64. 3 103 0. 63 91 33. 5 93 63. 2 101 0. 56 97 33. 5 93 69. 8 96 TABLE 8 [Eficet of polymyxin on the growth and double-stranded RNA synthesis of E. coli infected with MU-9 bacteriophage in tryptone broth] Polyrnixin added at 100 g/ml.

Growth (dry weight) Double-stranded RNA Time of addition of penicillin G post- Percent of Percent of mg./g., Percent of infection, minutes Mg./m1. control MgJl. control cells control Not added (common..- 0.67 100 53.4 100 79.6 100 0.10 0 0 o 0 0.12 18 0.83 2 6.92 9 0.18 37 1.7 3 9. 12 0. as 57 1s. 0 34 47. 3 59 o. 52 7s 40. 0 74 77. 0 97 0. 53 79 43. 7 s2 s2. 5 104 What is claimed is: 5. The process according to claim 1 in which the an- 1. The process for obtaining double-stranded rlbonlltibiotic is added before about minutes post-infection. cleic acid which comprises adding Escherichia coli to a 6. The process according to claim 1 in which the antinutrient culture medium and permitting growth to occur, biotic is added at 45 to minutes post-infection in the then adding MU-9 mutant of M82 coliphage to infect 20 amount of 5 to 200 micrograms per ml. of broth. the growing E. coli, then adding an antibiotic at about 45 to about 120 minutes post-infection to inhibit the References Cited growth of the E and thefeby obtam an {ncreased Davidson et al.: Progress In Nucleic Acid 'Research productivity of DS-RNP re1at1ve to the E. c011 cells. d M k l Biology, vol. 6, p. 377-381 (1967).

p f accordmg to clalm 1 Whlch the c111 25 Field et al.: National Academy of Sciences, vol. 58, ture medlum Includes tryrtolw- No. 5, November 1967, p. 2102-2108.

3. The process according to claim 1 in which the culture medium is corn steep liquor. I ALVIN E. TANENHOLTZ, Primary Examiner 4. The process according to claim 1 in whlch the MU-9 mutant is added when the E. coli count is at least 30 US. Cl. X.R.

5 x 10 cells per ml. of broth. 7 -2, 1.5 

